As the initial and rapid host defense against pathogens, the innate immune system mediates an inflammatory response to infection using a set of germline-encoded pattern recognition receptors (PRRs) to recognize conserved pathogen-associated molecular patterns (PAMPs) that are invariant among diverse groups of microorganisms. Proinflammatory chemokines and cytokines are then stimulated to activate other genes and induce such diverse inflammatory responses as lymphocyte activation, leukocytosis, fever, hypotension, decreased appetite, and arthritis. The molecular components of the innate immune system of vertebrates, insects, and plants are remarkably conserved, and in mammals, the Toll-like receptors (TLRs) function as PRRs. The Drosophila Toll receptor is required for embryonic patterning and was later shown to have a key role in the detection of fungal infection and induction of the anti-fungal peptide drosomycin. A family of six TLRs has been found in mammals, and in the mouse, TLR2 and TLR4 mediate recognition of the bacterial cell wall components peptidoglycan and lipopolysaccharide, respectively (Akira et al., Nat. Immunol., 2001, 2, 675–680; Schnare et al., Curr. Biol., 2000, 10, 1139–1142; Takeuchi and Akira, Int. Immunopharmacol., 2001, 1, 625–635; Wang et al., Infect. Immun., 2001, 69, 2270–2276).
PAMPs elicit activation of an intracellular signaling cascade via TLRs similar to that which occurs upon binding of endogenous cytokine ligands such as those of the interleukin-1 (IL-1) family to the IL-1 receptor (IL-1R). Although the Toll-like and IL-1 receptor signaling pathways are distinct, because the receptors share sequence similarity in their cytoplasmic regions, downstream events in their signaling pathways are mediated by common molecular components. The binding of cytokines to IL-1R family receptors or the binding of bacterial lipopolysaccharide or peptidoglycan to a TLR both ultimately result in the activation of the transcription factor NF-κB and its translocation to the nucleus, where it induces transcription of various genes. The activation of NF-κB by IL-1R, TLR2 and TLR4 receptors requires a common set of signal transduction molecules including IL-1 receptor-associated kinases (IRAKs), MyD88, tumor necrosis factor receptor-associated kinase 6 (TRAF6), NF-κB-inducing kinase (NIK), and IκB kinases (IKKs) (Akira et al., Nat. Immunol., 2001, 2, 675–680; Takeuchi and Akira, Int. Immunopharmacol., 2001, 1, 625–635; Wang et al., Infect. Immun., 2001, 69, 2270–2276).
MyD88 (also known as myeloid differentiation primary response gene 88 and MYD88) was identified as a myelopoietic differentiation factor activated in murine M1 myeloid precursors following induction of terminal differentiation and growth arrest by IL-6. MyD88 is activated in the absence of protein synthesis following induction of MlD+ leukemic myeloblasts as well as normal myeloid precursors for differentiation by either conditioned medium or IL-6 (Lord et al., Oncogene, 1990, 5, 387–396). The full length human MyD88 cDNA was cloned and the gene was found to be constitutively expressed as a 2.6 kilobase mRNA species in many adult human tissues, and in monocyte, T-, B-, NK-, and dendritic cells (Hardiman et al., Oncogene, 1996, 13, 2467–2475). The murine MyD88 gene was later determined to be 53 amino acids longer than originally predicted and the human MyD88 gene, encoding a cDNA with 5 exons, was mapped to human chromosomal locus 3p21.3-p22 by fluorescence in situ hybridization (Bonnert et al., FEBS Lett., 1997, 402, 81–84).
The EXCO9 EST appears to be a splice variant of MyD88 that encodes a MyD88 protein with a truncated C-terminal domain, and DNA sequence analysis of the genomic region encoding human MyD88 revealed two potential translation start sites (Hardiman et al., Oncogene, 1996, 13, 2467–2475). Northern blot analysis revealed that human MyD88 mRNA is expressed as two hybridizing species, 1.6 and 3 kilobases in length, in a variety of tissues and cell lines (Bonnert et al., FEBS Lett., 1997, 402, 81–84).
The role of MyD88 has been investigated in knockout mice. MyD88-deficient mice have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1, and all IL-1- and IL-18-mediated functions examined in these mice were impaired (Adachi et al., Immunity, 1998, 9, 143–150). These MyD88-deficient mice also were shown to be have enhanced susceptibility to Staphlococcus aureus infection and proinflammatory cytokine secretion by peritoneal macrophages was completely abrogated (Takeuchi et al., J. Immunol., 2000, 165, 5392–5396).
Bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides can stimulate murine and human lymphocytes, leading to secretion of cytokines, expression of co-stimulatory molecules, and to an increase in antigen-presenting function. In addition to macrophages and other lymphocytes, dendritic cells (DCs) are also critical sentinels in antimicrobial responses. They show a highly potent ability, as antigen presenting cells, to stimulate naïve T-cell activation. Exposure to microbial components such as lipopolysaccaharide and CpG DNA can induce DC maturation, characterized by cytokine production, up-regulation of co-stimulatory molecules, and an increased ability to activate T-cells. DCs from MyD88-deficient mice were used to show that CpG oligodeoxynucleotide DNA-induced DC maturation is MyD88-dependent (Akira et al., J. Endotoxin Res., 2000, 6, 383–387).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MyD88 and to date, investigative strategies aimed at modulating MyD88 function have involved the use of inactive mutants.
The MyD88 protein has a modular structure with an approximately 90-amino acid N-terminal “death domain” (DD), so called for its involvement hetero- or homodimeric protein interactions in apoptotic pathways, as well as its C-terminal domain Toll domain related to the cytoplasmic region of IL-1R and TLRs. The presence of these two domains suggests that MyD88 serves to connect an IL-1R or TLR with a downstream signaling mediator. A series of human MyD88 deletion mutants have been constructed to investigate the interactions between MyD88 and receptors upstream or signaling molecules downstream in 293T human embryonic kidney cells. Overexpression of an N-terminal deletion construct of MyD88 which leaves only the C-terminal region bearing homology to IL-1R acts as a dominant negative mutant, attenuating IL-1R-mediated NF-κB activation. Overexpression of a truncated version of MyD88 encoding only the death domain activates NF-κB. These and similar deletion constructs have allowed the molecular ordering of signaling components in this pathway, and it was determined that MyD88 functions upstream of TRAF6 (Medzhitov et al., Mol. Cell, 1998, 2, 253–258; Muzio et al., Science, 1997, 278, 1612–1615).
Many signaling pathways involving DD-containing adaptor proteins lead to activation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase pathway. Another series of death domain deletion mutations was generated as well as a point mutation which prevents dimerization of the death domain, and overexpression of these mutant constructs in 293T cells was found to inhibit IL-1 induced activation of NF-κB and JNK, suggesting that the death domain of MyD88 is critical for this activation (Burns et al., J. Biol. Chem., 1998, 273, 12203–12209).
The cytokines IL-1β, interferon gamma (IFNγ), and TNF-α are believed to play an important role in the destruction of insulin-producing β cells of the islets of Langerhans observed in type I diabetes. In particular, cytokine-induced production of nitric oxide has been shown to correlate with β cell apoptosis and/or inhibition of insulin secretion. β cells overexpressing dominant negative MyD88 death domain point and deletion mutants had an attenuated level of IL-1β/IFNγ-induced nitric oxide generation, an increased resistance to apoptosis, and maintained their insulin secretory response to glucose, indicating that MyD88 mediates the cytokine-induced apoptosis pathway leading to diabetes (Dupraz et al., J. Biol. Chem., 2000, 275, 37672–37678).
Deletion mutants have further established the position of MyD88 in the innate immune signaling pathway serving as a host defense against pathogens. Bacterial lipopolysaccharide is a potent activator of macrophages, eliciting rapid morphological changes such as cell spreading, which is mediated by cell-matrix proteins. Macrophage spreading is dependent on the p38 MAP kinase pathway and cytokine production. Furthermore, the induction of spreading was inhibited by overexpression of a dominant negative MyD88 deletion mutant, indicating that spreading of macrophages is mediated by MyD88 (Schmidt et al., Mol. Cell. Biol., 2001, 21, 438–448).
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of MyD88 expression.
The present invention provides compositions and methods for modulating MyD88 expression, including modulation of the alternative transcripts.